Targeting danger molecules in tendinopathy: the HMGB1/TLR4 axis

Abstract

ObjectivesTo seek evidence of the danger molecule, high-mobility group protein B1 (HMGB1) expression in human tendinopathy and thereafter, to explore mechanisms where HMGB1 may regulate inflammatory mediators and matrix regulation in human tendinopathy.MethodsTorn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing ‘early pathology’) biopsies were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from patients undergoing arthroscopic stabilisation surgery. Markers of inflammation and HMGB1 were quantified by reverse transcriptase PCR (RT-PCR) and immunohistochemistry. Human tendon-derived primary cells were derived from hamstring tendon tissue obtained during hamstring tendon anterior cruciate ligament reconstruction and used through passage 3. In vitro effects of recombinant HMGB1 on tenocyte matrix and inflammatory potential were measured using quantitative RT-PCR, ELISA and immunohistochemistry staining.ResultsTendinopathic tissues demonstrated significantly increased levels of the danger molecule HMGB1 compared with control tissues with early tendinopathy tissue showing the greatest expression. The addition of recombinant human HMGB1 to tenocytes led to significant increase in expression of a number of inflammatory mediators, including interleukin 1 beta (IL-1β), IL-6, IL-33, CCL2 and CXCL12, in vitro. Further analysis demonstrated rhHMGB1 treatment resulted in increased expression of genes involved in matrix remodelling. Significant increases were observed in Col3, Tenascin-C and Decorin. Moreover, blocking HMGB1 signalling via toll-like receptor 4 (TLR4) silencing reversed these key inflammatory and matrix changes.ConclusionHMGB1 is present in human tendinopathy and can regulate inflammatory cytokines and matrix changes. We propose HMGB1 as a mediator driving the inflammatory/matrix crosstalk and manipulation of the HMGB1/TLR4 axis may offer novel therapeutic approaches targeting inflammatory mechanisms in the management of human tendon disorders.

HMGB1 mediates the development of tendinopathy due to mechanical overloading

“…The tissue and cells derived from tendinopathic and ruptured Achilles tendons show evidence of chronic (non-resolving) inflammation (13). Additional clinical studies support this finding showing enhanced levels of HMGB1 in early stage supraspinatus tendinopathy tissues compared to normal tissues and late stage tendinopathy tissues (37, 38). Moreover, the in vitro study shows that recombinant HMGB1 induces significant inflammatory mediators such as IL-1β, IL-6, IL-33, CCL2, and CXCL-12 (37).…”

“…Additional clinical studies support this finding showing enhanced levels of HMGB1 in early stage supraspinatus tendinopathy tissues compared to normal tissues and late stage tendinopathy tissues (37, 38). Moreover, the in vitro study shows that recombinant HMGB1 induces significant inflammatory mediators such as IL-1β, IL-6, IL-33, CCL2, and CXCL-12 (37). The findings of this study further links HMGB1 with the inflammatory responses induced by mechanical overloading of tendon to the developmental course of tendinopathy.…”

“…Moreover, the amplification of the PGE 2 biosynthesis pathway by HMGB1/IL-1β is suggested as an important pathogenic mechanism perpetuating inflammatory and destructive activities in rheumatoid arthritis (51). While it is currently unclear whether our findings will reflect the actual mechanisms of tendinopathy development and treatment in humans, recent studies demonstrated that HMGB1 is present in human tendinopathic tendons and regulates cellular inflammation and protein production in vitro (37, 38). This suggests that HMGB1 plays a similar role in the development of tendinopathy due to mechanical overloading.…”

“…Previously, we have shown that excessive mechanical loading induces inflammatory mediator PGE 2 in tendon cells and tissues (15, 17). A few other studies have shown that upregulation of HMGB1 is associated with shoulder tendon injury/tendinopathy in patients and indicated that it would be a valuable target for tendinopathy management (37–39, 47). In this study, we extend these investigations to examine the role of HMGB1, an inflammatory alarmin molecule, in tendinopathy development due to mechanical overloading conditions.…”

Expression of alarmins in a murine rotator cuff tendinopathy model

“…Extracellular HMGB1 affects synovial inflammation and promotes macrophage and neutrophil-dependent development of arthritis 36,37. As Akbar et al38 reported, HMGB1 can regulate inflammatory cytokines and matrix changes through toll-like receptor 4 (TLR4) signaling in human tendinopathy. HMGB1 may play a role in the development of chronic inflammation in RC tendinopathy by regulating inflammatory cytokines and matrix changes.There was increased staining for IL-33 in the 1w, 4w, and 6w impingement groups(Figure 3).…”